Cinnamyl Alcohol Dehydrogenase Gene Regulates Bursaphelenchus xylophilus Reproduction and Development
, Wenjun Deng, Yu Zhang, Hongzheng Tai, Chao Wang, Guicai Du * and Ronggui Li *
Round 1
Reviewer 1 Report (Previous Reviewer 2)
The manuscript describes the results of a number of bioassays on pine wilt nematode to test the function of a gene that, after additional study, be used as a target for new pesticide research. Through down regulating the expression of the particular gene, the researchers noted that the a number of behavioral, developmental, reproductive behaviors together with virulence and susceptibility to ethanol were affected. The paper is clearly written, though some methods could be expanded on a little despite citations (or at least descriptions moved from results to methods in 1 or 2 cases, see below). The post-hoc test should be reported together with whether assumptions of t-test and ANOVA were met.
Specific comments:
Ln 33: revise to ‘droop’
Ln 35: remove . before citation
Ln 44: revise to ‘occurs’
Ln 76: revise to ‘understanding of’
Ln 78: revise to ‘provide a new’
Ln 221-222: revise for grammar. Consider revising and breaking sentences into 2. Potential revision could be something like: Eggs were separated and incubated in the dark at … The hatching rate was calculated as the as the number of hatched eggs divided by the total eggs at the start of the experiment. The sentence from results (Lns 417-418) could be placed here instead of the results.
Lns 235-237: Revise for grammar. Consider revising to: “A small wound was made at the top of the seeding and B. xylophilus were inoculated into the wound [56]. The growth of the Pinus thurnbergii seedlings were observed every day [56].” What growth metric was measured? Height? Radial growth at root collar? And report that seedling mortality was recorded too. And add the measurement intervals, which are reported later in the results Ln 431.
Lns 237-248: Use genus abbreviation.
Ln 244: Check with journal style guide on how numbers are reported. But generally because of the placement of “ten thousand” in this sentence, it can be written out as a number: 10,000. Same with other places where the number does not start the sentence and it is equal to or greater than 10 (e.g., Ln 253 should be “One hundred µL … 300 B. xylophilus…”).
Table 2 and Figure 9 captions: define abbreviations of SOD, POD, and CAT
Ln 517-518: Revise to “PWD include”. Is “breeding PWN-infected pine trees” mean developing a PWD resistance breeding program? How does breeding PWN-infected pine trees prevent disease?
Author Response
The authors of this article sincerely thank the reviewers for their useful comments and for the time spent on suggesting for revising this manuscript. All the reviewers’ suggestions have been carefully considered, and we’ve tried our best to improve the manuscript as corresponded corrections have been added to the revised manuscript. This text was prepared to present our point-by-point responses to the reviewers’ comments and to clarify how we addressed them during the revision. All modifications are highlighted in yellow (according to Reviewer 1’ comments), blue (according to Reviewer 2’ comments) and green (according to suggestions from a colleague fluent in English writing) in the manuscript.
Point 1: Ln 33: revise to ‘droop’.
Response 1: Ln 33: ‘drooped’ has been revised to ‘droop’.
Point 2: Ln 35: remove . before citation.
Response 2: Ln 35: we have removed ‘.’in front of the citation [6-7].
Point 3: Ln 44: revise to ‘occurs’.
Response 3: Ln 44: ‘occurred’ has been revised to ‘occurs’.
Point 4: Ln 76: revise to ‘understanding of’.
Response 4: Ln 76: ‘understand the roles of’ has been revised to ‘understanding of’.
Point 5: Ln 78: revise to ‘provide a new’.
Response 5: Ln 78: ‘provide new’ has been revised to ‘provide a new’.
Point 6: Ln 221-222: revise for grammar. Consider revising and breaking sentences into 2. Potential revision could be something like: Eggs were separated and incubated in the dark at … The hatching rate was calculated as the as the number of hatched eggs divided by the total eggs at the start of the experiment. The sentence from results (Lns 417-418) could be placed here instead of the results.
Response 6: Lns 221-222: “Separate the eggs and incubated in the dark at 25 °C for 24 h for hatching, and the hatching rate was calculated.” has been revised to “Eggs were separated and incubated in the dark at 25 °C for 24 h for hatching. The number of total eggs at the beginning of assay and the remaining eggs after 24 h was recorded to calculate the number of hatched eggs and the hatching rate.” Lns 417-418: “The number of total eggs at the beginning of assay and the remaining eggs after 24 h was recorded to calculate the number of hatched eggs and the hatching rate.” has been deleted.
Point 7: Lns 235-237: Revise for grammar. Consider revising to: “A small wound was made at the top of the seeding and B. xylophilus were inoculated into the wound [56]. The growth of the Pinus thurnbergii seedlings were observed every day [56].” What growth metric was measured? Height? Radial growth at root collar? And report that seedling mortality was recorded too. And add the measurement intervals, which are reported later in the results Ln 431.
Response 7: Lns 235-237: “A small wound was made at the top of the seedling, and inoculated the B. xylophilus were into the wound, observe the growth of the Pinus thunbergii seedlings every day [56]” revise to “A small wound was made at the top of the seeding and B. xylophilus were inoculated into the wound [56]. The wilting of pine needles of the Pinus thurnbergii seedlings were observed every day [56]. And the seedling mortality was recorded at 0, 9 and 21 days, respectively.”
Point 8: Lns 237-248: Use genus abbreviation.
Response 8: Lns 237-248: ‘Bursaphelenchus xylophilus’ has been revised to ‘B. xylophilus’, ‘Botrytis cinerea’ revise to ‘B. cinerea’.
Point 9: Ln 244: Check with journal style guide on how numbers are reported. But generally because of the placement of “ten thousand” in this sentence, it can be written out as a number: 10,000. Same with other places where the number does not start the sentence and it is equal to or greater than 10 (e.g., Ln 253 should be “One hundred µL … 300 B. xylophilus…”).
Response 9: Ln 188: ‘three thousand’ has been revised to ‘3,000’. Ln 203: ‘three thousand’ has been revised to ‘3,000’. Ln 211: ‘two hundred’ has been revised to ‘200’. Ln 219: “100 μL of B. xylophilus suspension (approximately four hundred B. xylophilus with ratio of female to male of 1:1)” has been revised to “One hundred microliters of B. xylophilus suspension (approximately 400 B. xylophilus with ratio of female to male of 1:1)”. Ln 227: “100 μL of B. xylophilus suspension (approximately two hundred mixed developmen-tal stages B. xylophilus)” has been revised to “One hundred microliters of B. xylophilus suspension (approximately 200 mixed develop-mental stages B. xylophilus)”. Ln 234: ‘two hundred’ has been revised to ‘200’. Ln 244: ‘ten thousand’ has been revised to ‘10,000’. Ln 248: ‘three thousand’ has been revised to ‘3,000’. Ln 253: “100 μL of B. xylophilus suspension (approximately three hundred B. xylophilus with ratio of female to male of 1:1)” has been revised to “One hundred microliters of B. xylophilus suspension (approximately 300 B. xylophilus with ratio of female to male of 1:1)”. Ln 259: ‘three thousand’ has been revised to ‘3,000’.
Point 10: Table 2 and Figure 9 captions: define abbreviations of SOD, POD, and CAT.
Response 10: We have changed the caption of Table 3 to “Table 3 Formulas used for calculating superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities of B. xylophilus”. Changed the caption of Figure 9 (d) to “(d), The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) of B. xylophilus after RNAi.”.
Point 11: Ln 517-518: Revise to “PWD include”. Is “breeding PWN-infected pine trees” mean developing a PWD resistance breeding program? How does breeding PWN-infected pine trees prevent disease?
Response 11: Ln 517: ‘PWD at include’ has been revised to ‘PWD include’. Ln 518: ‘PWN-infected’ has been revised to ‘PWN-resistant’. We intended to breed pine trees that are resistant to PWD. Here is an spelling error.
Reviewer 2 Report (New Reviewer)
Overall, I think this is an interesting and soundly conducted study. The authors have presented a strong case for the role of cinnamyl alcohol dehydrogenase gene in PWN. They have also presented evidence that this gene is a candidate for targeting pesticide development for PWN and PWD. I believe this paper only requires moderate editing before being publication ready.
A few initial thoughts I have are as follows. What species of pines are susceptible to PWN/PWD? If you do not wish to list all of the hosts, I recommend at least highlighting the major species threatened by this disease in Asia. Do we know how PWN was first introduced into China? If not, any proposed pathways? What is the established range in Asia? How fast is it spreading per year? I think giving a little more background on PWN/PWD and the threat, both ecological and economical, it poses in China/Asia.
The following are my specific edits/recommendations:
Line 8: global should be globally distributed
Line 12: this is the first mention so E. coli should be Escherichia coli
Line 17: I recommend removing “ability” and stating “the movement, feeding, reproduction,…”
Line 21: This study revealed a promising target for PWD control.
Line 27: has the characteristics of should be “is characterized by”
Line 29-30: add “and other cerambycid beetles.” After Monochamus alteratus
Line 31: remove “their physiological activities start to change, which included” and replace with …PWD causes a decrease of resin…
Line 32: Remove “At the middle stage of infection,” and replace with “As the disease progresses, needles turn yellowish…”
Line 46: Change “pine trees with infection of B. xylophilus and block water transport” with “in B. xylophilus infected pine trees and blocks water transport.”
Line 48: change “for pine” to “in pine” and “while its symbiosis” to “but the symbiosis”
Line 68-69: Change “and the migration rate of B. xylophilus slowed down in pine trees” to “and effectively slowed B. xylophilus migration.” Where was this work done? Consider stating that here.
Line 70-71: Break this sentence up. I suggest ending the first sentence after “…virulent strain.” And then “Silencing this gene significantly reduced B. xylophilus virulence.”
Line 72-73: Remove “which can effectively inhibit the activity of alcohol dehydrogenase” as this is redundant.
Lines 75-78: Expand on exactly what this study was designed to address. Consider including specific hypotheses.
Line 82: How exactly were PWN collected? This isn’t ever clearly stated.
Lines 83-98: This is essentially a large list of products/technologies used in your experiments. Consider putting this information into a table instead of a paragraph.
Line 99: remove “Cultivation of Pinus thunberii seedlings: the” and start the sentence with “Mature cones…”
Line 109: Approximate should be Approximately
Line 129: inputted should be entered
Line 132: engineering should be engineered
Line 199: add “with” between performed and three
Line 202: change motor ability to movement (do this here and throughout)
Line 216: Change Five repeats were performed to The experiment was repeated five times. Do this here and throughout (i.e., line 224, 231, etc)
Line 221: Separate the eggs should be Eggs were separated…
Line 229: Change “survival number was counted” to “the number of surviving B. xylophilus were counted…”
Line 318: Change “higher” to something like “strong”. It is awkward to use higher after you mentioned a greater percentage in the previous sentence…
Figure 5: Consider adding letters to indicate significant differences.
Line 367-368: Split this into two sentences. Add a period after decreased and start a new sentence with “Fragment 2…”
Line 380: Change to “B. xylophilus movement and reproduction”
Line 381-384: This in information that belongs in the Methods
Lines 414 and 416: Change significantly different from to significantly lower than in both cases. Be specific and indicate the direction of difference when you highlight specific results in the text. Please review this throughout the paper.
Figure 7d: Are the letters of statistical significance correct in this figure? It seems odd to me that 4-methylpyrozole is not different from Fragment 3…
Line 426: approximate should be approximately
Line 516: change to “severe damage to pine forests.”
Line 517-518: What do you mean by “breeding PWN-infected pine trees”? Do you mean breeding PWN-resistant?
Line 519: change chemical drugs to pesticides
Line 521: Change drugs to pesticides
Line 522: delete the first sentence of this paragraph.
Line 524: add “an” between of and infected
Line 531: Besides should be Additionally
Line 532: Italicize “in situ”
Line 541: Change to “Therefore, further study of BxCAS-1 is warranted.”
Line 542-543: Delete “is mainly studied in plants and microorganisms, which”
Line 559: delete “execution”
Line 566: change explosively to dramatically
Line 573: Change Besides to Additionally,
The quality of English language in this manuscript is decent; however, does require moderate editing. I have highlighted most of the issues in my specific comments above. I recommend additional review once my suggestions have been implemented.
Author Response
The authors of this article sincerely thank the reviewers for their useful comments and for the time spent on suggesting for revising this manuscript. All the reviewers’ suggestions have been carefully considered, and we’ve tried our best to improve the manuscript as corresponded corrections have been added to the revised manuscript. This text was prepared to present our point-by-point responses to the reviewers’ comments and to clarify how we addressed them during the revision. All modifications are highlighted in yellow (according to Reviewer 1’ comments), blue (according to Reviewer 2’ comments) and green (according to suggestions from a colleague fluent in English writing) in the manuscript.
Point 1: Line 8: global should be globally distributed.
Response 1: Line 8: ‘global’ has been revised to ‘globally distributed’.
Point 2: Line 12: this is the first mention so E. coli should be Escherichia coli.
Response 2: Line 12: ‘E. coli’ has been revised to ‘Escherichia coli’.
Point 3: Line 17: I recommend removing “ability” and stating “the movement, feeding, reproduction,…”.
Response 3: Line 17: “movement ability, feeding ability, reproductive ability” has been revised to “movement, feeding, reproduction”.
Point 4: Line 21: This study revealed a promising target for PWD control.
Response 4: Line 21: “This study offered a promising target for new pesticides research in order to control PWD.” has been revised to “This study revealed a promising target for PWD control.”.
Point 5: Line 27: has the characteristics of should be “is characterized by”.
Response 5: Line 27: “has the characteristics of” has been revised to “is characterized by”.
Point 6: Line 29-30: add “and other cerambycid beetles.” After Monochamus alteratus.
Response 6: Line 29-30: we have added “and other cerambycid beetles.” behind Monochamus alteratus.
Point 7: Line 31: remove “their physiological activities start to change, which included” and replace with …PWD causes a decrease of resin…
Response 7: Line 31: “their physiological activities start to change, which included decrease of resin secretion,” has been revised to “PWD causes a decrease of resin secretion,”.
Point 8: Line 32: Remove “At the middle stage of infection,” and replace with “As the disease progresses, needles turn yellowish…”
Response 8: Line 32: “At the middle stage of infection,” has been revised to “As the disease progresses,”.
Point 9: Line 46: Change “pine trees with infection of B. xylophilus and block water transport” with “in B. xylophilus infected pine trees and blocks water transport.”
Response 9: Line 46: “pine trees with infection of B. xylophilus” has been revised to “B. xylophilus infected pine trees”.
Point 10: Line 48: change “for pine” to “in pine” and “while its symbiosis” to “but the symbiosis”
Response 10: Line 48: ‘for pine’ has been revised to ‘in pine’, and ‘while its symbiosis’ has been revised to ‘but the symbiosis’.
Point 11: Line 68-69: Change “and the migration rate of B. xylophilus slowed down in pine trees” to “and effectively slowed B. xylophilus migration.” Where was this work done? Consider stating that here.
Response 11: Line 68-69: “and the migration rate of B. xylophilus slowed down in pine trees.” has been revised to “and effectively slowed B. xylophilus migration.”.
Point 12: Line 70-71: Break this sentence up. I suggest ending the first sentence after “…virulent strain.” And then “Silencing this gene significantly reduced B. xylophilus virulence.”
Response 12: Line 70-71: “was upregulated in the highly virulent strain, and the virulence of B. xylophilus was significantly reduced by silencing this gene.” has been revised to “was upregulated in the highly virulent strain. Silencing this gene significantly reduced B. xylophilus virulence.”
Point 13: Line 72-73: Remove “which can effectively inhibit the activity of alcohol dehydrogenase” as this is redundant.
Response 13: Line 72-73: we have removed “which can effectively inhibit the activity of alcohol dehydrogenase”.
Point 14: Lines 75-78: Expand on exactly what this study was designed to address. Consider including specific hypotheses.
Response 14: Lines 75-78: we have changed to “At present, there are few studies on cinnamyl alcohol dehydrogenase in animals, and cinnamyl alcohol dehydrogenase has never been reported B. xylophilus. The data from this study will support the understanding of BxCAD-1 in the reproduction and development of B. xylophilus, which may be an important pathogenic factor of B. xylophilus and provide a new molecular target for exploration of nematicides and control of PWD. If it is universal, it can provide some data support for the control of other nematodes.”
Point 15: Line 82: How exactly were PWN collected? This isn’t ever clearly stated.
Response 15: Line 82: The method and reference of collecting PWN were added.
Point 16: Lines 83-98: This is essentially a large list of products/technologies used in your experiments. Consider putting this information into a table instead of a paragraph.
Response 16: Lines 83-98: The text description has been changed to table form.
Point 17: Line 99: remove “Cultivation of Pinus thunberii seedlings: the” and start the sentence with “Mature cones…”
Response 17: Line 99: “Cultivation of Pinus thunbergii seedlings: the mature cones of Pinus thunbergii on the campus of Qingdao University were collected,” has been revised to “Mature cones of Pinus thunbergii on the campus of Qingdao University were collected,”.
Point 18: Line 109: Approximate should be Approximately.
Response 18: Line 109: ‘Approximate’ has been revised to ‘Approximately’.
Point 19: Line 129: inputted should be entered.
Response 19: Line 129: ‘inputted’ has been revised to ‘entered’.
Point 20: Line 132: engineering should be engineered.
Response 20: Line 132: ‘engineering’ has been revised to ‘engineered’.
Point 21: Line 199: add “with” between performed and three.
Response 21: Line 199: ‘performed three’ has been revised to ‘performed with three’.
Point 22: Line 202: change motor ability to movement (do this here and throughout).
Response 22: Line 202: “Effect of RNAi on motor ability” has been revised to “Effect of RNAi on movement”. And the caption of Figure 7 to “Effects of RNAi on movement (a)”.
Point 23: Line 216: Change Five repeats were performed to The experiment was repeated five times. Do this here and throughout (i.e., line 224, 231, etc).
Response 23: Line 216: “Five repeats were performed.” has been revised to “The experiment was repeated five times.”. Line 224: “Three repeats were performed.” has been revised to “The experiment was repeated three times.”. Line 231: “Three repeats were performed.” has been revised to “The experiment was repeated three times.”. Line 240: “Eight repeats were performed.” has been revised to “The experiment was repeated eight times.”. Line 251: “Three repeats were performed.” has been revised to “The experiment was repeated three times.”. Line 257: “Three repeats were performed.” has been revised to “The experiment was repeated three times.”. Line 269: “Three repeats were performed.” has been revised to “The experiment was repeated three times.”. Line 280: “Three repeats were performed.” has been revised to “The experiment was repeated three times.”.
Point 24: Line 221: Separate the eggs should be Eggs were separated…
Response 24: Line 221: “Separate the eggs” has been revised to “Eggs were separated”.
Point 25: Line 229: Change “survival number was counted” to “the number of surviving B. xylophilus were counted…”
Response 25: Line 229: “survival number was counted” has been revised to “the number of surviving B. xylophilus were counted”.
Point 26: Line 318: Change “higher” to something like “strong”. It is awkward to use higher after you mentioned a greater percentage in the previous sentence…
Response 26: Line 318: ‘higher’ has been revised to ’strong’.
Point 27: Figure 5: Consider adding letters to indicate significant differences.
Response 27: In Figure 5, different letters have been used to indicate significant differences among groups.
Point 28: Line 367-368: Split this into two sentences. Add a period after decreased and start a new sentence with “Fragment 2…”.
Response 28: Line 367-368: “that in the B. xylophilus soaked in fragment 1, 2 and 3 dsRNA solution decreased, with fragment 2 had the greatest impact on gene expression level.” has been revised to “that in the B. xylophilus soaked in fragment 1, 2 and 3 dsRNA solution decreased. Fragment 2 had the greatest impact on gene expression level.”.
Point 29: Line 380: Change to “B. xylophilus movement and reproduction”
Response 29: Line 380: “Motor and reproduction ability of B. xylophilus” has been revised to “B. xylophilus movement and reproduction”.
Point 30: Line 381-384: This in information that belongs in the Methods
Response 30: Line 381-384: we have deleted “Bursaphelenchus xylophilus was treated with interfering fragment 1, fragment 2, fragment 3, using nematodes treated with distilled H2O and dsRNA derived from gfp as negative control, and those treated with 4-methylpyrazole as positive control. The head thrashes of B. xylophilus treated differently in 1 min were counted.”
Point 31: Lines 414 and 416: Change significantly different from to significantly lower than in both cases. Be specific and indicate the direction of difference when you highlight specific results in the text. Please review this throughout the paper.
Response 31: Line 414: ‘significantly different’ has been revised to ‘significantly lower’. Line 416: ‘significantly different’ has been revised to ‘significantly lower’. Line 421: ‘significantly different’ was revised to ‘significantly lower’. Line 488: ‘significantly different’ has been revised to ‘significantly lower’.
Point 32: Figure 7d: Are the letters of statistical significance correct in this figure? It seems odd to me that 4-methylpyrozole is not different from Fragment 3…
Response 32: The markers of 4-methylpyrazole group and fragment 3 group in Figure 7d were e and c, respectively. The figure has been updated.
Point 33: Line 426: approximate should be approximately.
Response 33: Line 426: ‘approximate’ has been revised to ‘approximately’.
Point 34: Line 516: change to “severe damage to pine forests.”
Response 34: Line 516: ‘severe damages to forests’ has been revised to ‘severe damages to pine forests’.
Point 35: Line 517-518: What do you mean by “breeding PWN-infected pine trees”? Do you mean breeding PWN-resistant?
Response 35: Line 517-518: ‘PWN-infected’ has been revised to ‘PWN-resistant’. We intended to describe pine trees that are resistant to PWD. Here is a spelling mistake.
Point 36: Line 519: change chemical drugs to pesticides.
Response 36: Line 519: ‘chemical drugs’ has been revised to ‘pesticides’.
Point 37: Line 521: Change drugs to pesticides.
Response 37: Line 521: ‘drugs’ was revised to ‘pesticides’.
Point 38: Line 522: delete the first sentence of this paragraph.
Response 38: Line 522: we have deleted “In order to further clarify the pathogenic mechanism of PWN, molecular biology techniques were used to study the pathogenic genes of PWN.”.
Point 39: Line 524: add “an” between of and infected.
Response 39: Line 524: ‘of infected’ has been revised to ‘of an infected’.
Point 40: Line 531: Besides should be Additionally.
Response 40: Line 531: ‘Besides’ has been revised to ‘Additionally’.
Point 41: Line 532: Italicize “in situ”.
Response 41: Line 532: Italicize “in situ”.
Point 42: Line 541: Change to “Therefore, further study of BxCAS-1 is warranted.”
Response 42: Line 541: “Therefore, we will further study BxCAD-1.” has been revised to “Therefore, further study of BxCAS-1 is warranted.”.
Point 43: Line 542-543: Delete “is mainly studied in plants and microorganisms, which”.
Response 43: Line 542-543: “The cinnamyl alcohol dehydrogenase is mainly studied in plants and microorgan- 542 isms, which is mainly involved in the formation of lignin in plants, but plays other roles 543 in microorganisms [28-37].” was revise to “The cinnamyl alcohol dehydrogenase is mainly involved in the formation of lignin in plants, but plays other roles in microorganisms [28-37].”
Point 44: Line 559: delete “execution”.
Response 44: Line 559: we have deleted “execution”.
Point 45: Line 566: change explosively to dramatically.
Response 45: Line 566: ‘explosively’ has been revised to ‘dramatically’.
Point 46: Line 573: Change Besides to Additionally.
Response 46: Line 573: ‘Besides’ was
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript “A gene coding cinnamyl alcohol dehydrogenase regulates reproduction and development of Bursaphelenchus xylophilus” presents the characterisation of a species-specific B. xylophilus protein (cinnamyl alcohol dehydrogenase) and the phenotypic effects of RNA interference of its coding gene. The manuscript indicates some important information related with pinewood nematode pathogenicity and it is in the scope of the journal. However, there are some important issues that are not presented correctly, and, at this stage, this manuscript is not suitable for publication. Main concerns are related with the methodology used for testing the RNAi efficiency and the corresponding phenotypic effects. The methodologies used are not well explicated and confusing and have flaws; the controls are not clear, there no reference to the statistical test used and in consequence the results are not well presented and are unclear. There is lack of information in the introduction and in the discussion, no comparisons are made with previous RNAi results obtained with of other Bx genes (efficiency and phenotipic effects). Comments and suggestions are made in the attached pdf file.
Comments for author File: 
Comments.pdf
The manuscript need a moderate English language editing.
Reviewer 2 Report
Manuscript describes the results of a number of bioassays to assess reproductive and developmental affects on B. xylophilus by down regulation of a gene coding for cinnamyl alcohol dehydrogenase. The manuscript is well written and the results were well described. The statistical tests used needs to be reported. The conclusions mainly fit the results with the caveat that additional investigations need to occur.
Specific comments:
Lns 32-36, 42-52: Revise to present tense
Lns 36-37: Revise sentence and correct to ‘severe’. Consider revision: Pine trees in North America, where PWD originated, are not severely affected.
Ln 63: Use scientific names
Ln 89: Italicize Botrytis cinereal
Ln 114: Revise to ‘tree of’
Lns 251-252: Need more detail on statistical tests used and how assumptions were met.
Ln 293: Revise to “highest catalytic”
Lns 293-297, Figure 5: What are the quantitative statistics on these data to say that certain treatments had the highest or lowest activity? Need to conduct statistics with pos hoc test to make these statements.
Figure 6: What statistical test and post hoc tests were performed?
Ln 360: Revise to “B. xylophilus treated”
Lns 369-370: Revise for clarity. I don’t understand what “with for 4.67” means.
Figure 6, 8: Define what different letters and asterisk mean.
Ln 441: Revise to “BxCAD-1 with”
Ln 466; Revise to “low concentrations of ethanol”
Ln 467: Remove hyphen
Ln 476: Revise to “appear to have clumping”
